Polypeptides having endoglucanase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having endoglucanase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/918,452 filed on Nov. 22, 2010, which is a 35 U.S.C. 371 nationalapplication of PCT/US2009/36316 filed on Mar. 6, 2009, which claimspriority or the benefit under 35 U.S.C. 119 of U.S. ProvisionalApplication No. 61/034,405 filed on Mar. 6, 2008, the contents of whichare fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingendoglucanase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods of producing and using the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose linked by beta-1,4bonds. Many microorganisms produce enzymes that hydrolyze beta-linkedglucans. These enzymes include endoglucanases, cellobiohydrolases, andbeta-glucosidases. Endoglucanases digest the cellulose polymer at randomlocations, opening it to attack by cellobiohydrolases.Cellobiohydrolases sequentially release molecules of cellobiose from theends of the cellulose polymer. Cellobiohydrolase I is a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) activity whichcatalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellotetriose, or any beta-1,4-linked glucose containing polymer,releasing cellobiose from the reducing ends of the chain.Cellobiohydrolase II is a 1,4-D-glucan cellobiohydrolase (E.C. 3.2.1.91)activity which catalyzes the hydrolysis of 1,4-beta-D-glucosidiclinkages in cellulose, cellotetriose, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the non-reducing ends ofthe chain. Cellobiose is a water-soluble beta-1,4-linked dimer ofglucose. Beta-glucosidases hydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

Jørgensen et al., 2003, Enzyme and Microbial Technology 32: 851-861,Thygesen et al., 2003, Enzyme and Microbial Technology 32: 606-615, andJørgensen and Olsson, 2006, Enzyme and Microbial Technology 38: 381-390disclose cellulose-degrading enzymes from Penicillium brasilianum IBT20888.

It would be an advantage in the art to identify new endoglucanaseshaving improved properties, such as improved hydrolysis rate, betterthermal stability, reduced adsorption to lignin, and/or ability tohydrolyze non-cellulosic components of biomass, such as hemicellulose,in addition to hydrolyzing cellulose. Endoglucanases with a broad rangeof side activities on hemicellulose can be especially beneficial forimproving the overall hydrolysis yield of complex, hemicellulose-richbiomass substrates.

The present invention provides polypeptides having endoglucanaseactivity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingendoglucanase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence comprising a partialamino acid sequence having at least 75% identity to the partial aminoacid sequence of SEQ ID NO: 2 or at least 85% identity to the partialamino acid sequence of SEQ ID NO: 4;

(b) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence comprising a partial nucleotide sequence that hybridizes underat least high stringency conditions with (i) the partial nucleotidesequence of SEQ ID NO: 1 or the partial nucleotide sequence of SEQ IDNO: 3, (ii) the cDNA sequence contained in the partial nucleotidesequence of SEQ ID NO: 1 or the partial nucleotide sequence of SEQ IDNO: 3, or (iii) a full-length complementary strand of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence comprising a partial nucleotide sequence having at least 75%identity to the partial nucleotide sequence of SEQ ID NO: 1 or at least85% identity to the partial nucleotide sequence of SEQ ID NO: 3; and

(d) a variant comprising an amino acid sequence comprising asubstitution, deletion, and/or insertion of one or more (several) aminoacids of the partial amino acid sequence of SEQ ID NO: 2 or the partialamino acid sequence of SEQ ID NO: 4.

The present invention also relates to isolated polynucleotides encodingpolypeptides having endoglucanase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence comprising a partial amino acid sequence having at least 75%identity to the partial amino acid sequence of SEQ ID NO: 2 or at least85% identity to the partial amino acid sequence of SEQ ID NO: 4;

(b) a polynucleotide comprising a nucleotide sequence comprising apartial nucleotide sequence that hybridizes under at least highstringency conditions with (i) the partial nucleotide sequence of SEQ IDNO: 1 or the partial nucleotide sequence of SEQ ID NO: 3, (ii) the cDNAsequence contained in the partial nucleotide sequence of SEQ ID NO: 1 orthe partial nucleotide sequence of SEQ ID NO: 3, or (iii) a full-lengthcomplementary strand of (i) or (ii);

(c) a polynucleotide comprising a nucleotide sequence comprising apartial nucleotide sequence having at least 75% identity to the partialnucleotide sequence of SEQ ID NO: 1 or at least 85% identity to thepartial nucleotide sequence of SEQ ID NO: 3; and

(d) a polynucleotide encoding a variant comprising an amino acidsequence comprising a substitution, deletion, and/or insertion of one ormore (several) amino acids of the partial amino acid sequence of SEQ IDNO: 2 or the partial amino acid sequence of SEQ ID NO: 4.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing a polypeptide havingendoglucanase activity.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having endoglucanase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. The presentalso relates to such a double-stranded inhibitory RNA (dsRNA) molecule,wherein optionally the dsRNA is a siRNA or a miRNA molecule.

The present invention also relates to methods of using the polypeptideshaving endoglucanase activity in the conversion of cellulose to glucoseand various substances.

The present invention also relates to plants comprising an isolatedpolynucleotide encoding a polypeptide having endoglucanase activity.

The present invention also relates to methods of producing a polypeptidehaving endoglucanase activity, comprising: (a) cultivating a transgenicplant or a plant cell comprising a polynucleotide encoding thepolypeptide having endoglucanase activity under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the partial genomic DNA sequence and the deduced amino acidsequence of a Penicillium brasilianum IBT 20888 Family 5 endoglucanase(SEQ ID NOs: 1 and 2, respectively).

FIG. 2 shows the partial genomic DNA sequence and the deduced amino acidsequence of a Penicillium brasilianum IBT 20888 Family 5 endoglucanase(SEQ ID NOs: 3 and 4, respectively).

DEFINITIONS

Endoglucanase activity: The term “endoglucanase activity” is definedherein as an endo-1,4-beta-D-glucan 4-glucanohydrolase (E.C. No.3.2.1.4) that catalyses the endohydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (such as carboxymethylcellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixedbeta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and otherplant material containing cellulosic components. For purposes of thepresent invention, endoglucanase activity is determined usingcarboxymethyl cellulose (CMC) hydrolysis according to the procedure ofGhose, 1987, Pure and Appl. Chem. 59: 257-268. One unit of endoglucanaseactivity is defined as 1.0 μmole of reducing sugars produced per minuteat 50° C., pH 4.8.

The polypeptides of the present invention having endoglucanase activitymay further have enzyme activity toward one or more substrates selectedfrom the group consisting of xylan, xyloglucan, arabinoxylan,1,4-beta-D-mannan, and galactomannan. The activity of the polypeptideshaving endoglucanase activity on these polysaccharide substrates isdetermined as percent of the substrate hydrolyzed to reducing sugarsafter incubating the substrate (5 mg per ml) with a polypeptide havingendoglucanase activity of the present invention (5 mg protein per g ofsubstrate) for 24 hours with intermittent stirring at pH 5.0 (50 mMsodium acetate) and 50° C. Reducing sugars in hydrolysis mixtures aredetermined by the p-hydroxybenzoic acid hydrazide (PHBAH) assay.

In one aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity toward xylan. Inanother aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity toward xyloglucan.In another aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity toward arabinoxylan.In another aspect, the polypeptides of the present invention havingendoglucanase activity further have enzyme activity toward1,4-beta-D-mannan. In another aspect, the polypeptides of the presentinvention having endoglucanase activity further have enzyme activitytoward galactomannan. In another aspect, the polypeptides of the presentinvention having endoglucanase activity further have enzyme activitytoward xylan, xyloglucan, arabinoxylan, 1,4-beta-D-mannan, and/orgalactomannan.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the endoglucanase activity of thepolypeptide comprising an amino acid sequence comprising the partialamino acid sequence of SEQ ID NO: 2 or the partial amino acid sequenceof SEQ ID NO: 4.

Cellobiohydrolase: The term “cellobiohydrolase” is defined herein as a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes thehydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,cellooligosaccharides, or any beta-1,4-linked glucose containingpolymer, releasing cellobiose from the reducing or non-reducing ends ofthe chain. For purposes of the present invention, cellobiohydrolaseactivity is determined according to the procedures described by Lever etal., 1972, Anal. Biochem. 47: 273-279 and by van Tilbeurgh et al., 1982,FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBSLetters 187: 283-288. In the present invention, the Lever et al. methodwas employed to assess hydrolysis of cellulose in corn stover, while themethod of van Tilbeurgh et al. was used to determine thecellobiohydrolase activity on a fluorescent disaccharide derivative.

Beta-glucosidase: The term “beta-glucosidase” is defined herein as abeta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes thehydrolysis of terminal non-reducing beta-D-glucose residues with therelease of beta-D-glucose. For purposes of the present invention,beta-glucosidase activity is determined according to the basic proceduredescribed by Venturi et al., 2002, J. Basic Microbiol. 42: 55-66, exceptdifferent conditions were employed as described herein. One unit ofbeta-glucosidase activity is defined as 1.0 μmole of p-nitrophenolproduced per minute at 50° C., pH 5 from 4 mMp-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodiumcitrate, 0.01% TWEEN® 20.

Family 5 glycoside hydrolase or Family GH5 or GH5: The term “Family 5glycoside hydrolase” or “Family GH5” or “GH5” is defined herein as apolypeptide falling into the glycoside hydrolase Family 5 according toHenrissat B., 1991, A classification of glycoside hydrolases based onamino-acid sequence similarities, Biochem. J. 280: 309-316, andHenrissat B., and Bairoch A., 1996, Updating the sequence-basedclassification of glycoside hydrolases, Biochem. J. 316: 695-696.

Family 61 glycoside hydrolase or Family 61 or GH61: The term “Family 61glycoside hydrolase” or “Family GH61” or “GH61” is defined herein as apolypeptide falling into the glycoside hydrolase Family 61 according toHenrissat B., 1991, A classification of glycosyl hydrolases based onamino-acid sequence similarities, Biochem. J. 280: 309-316, andHenrissat B., and Bairoch A., 1996, Updating the sequence-basedclassification of glycosyl hydrolases, Biochem. J. 316: 695-696.Presently, Henrissat lists the GH61 Family as unclassified indicatingthat properties such as mechanism, catalytic nucleophile/base, andcatalytic proton donors are not known for polypeptides belonging to thisfamily.

Cellulosic material: The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant ishemi-cellulose, and the third is pectin. The secondary cell wall,produced after the cell has stopped growing, also containspolysaccharides and is strengthened by polymeric lignin covalentlycross-linked to hemicellulose. Cellulose is a homopolymer ofanhydrocellobiose and thus a linear beta-(1-4)-D-glucan, whilehemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

The cellulosic material can be any material containing cellulose.Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, herbaceous material,agricultural residue, forestry residue, municipal solid waste, wastepaper, and pulp and paper mill residue The cellulosic material can beany type of biomass including, but not limited to, wood resources,municipal solid waste, wastepaper, crops, and crop residues (see, forexample, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E.Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.; Wyman,1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistryand Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progressin Bioconversion of Lignocellulosics, in Advances in BiochemicalEngineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp.23-40, Springer-Verlag, New York). It is understood herein that thecellulose may be in the form of lignocellulose, a plant cell wallmaterial containing lignin, cellulose, and hemicellulose in a mixedmatrix.

In one aspect, the cellulosic material is herbaceous material. Inanother aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is forestry residue. In anotheraspect, the cellulosic material is municipal solid waste. In anotheraspect, the cellulosic material is waste paper. In another aspect, thecellulosic material is pulp and paper mill residue.

In another aspect, the cellulosic material is corn stover. In anotherpreferred aspect, the cellulosic material is corn fiber. In anotheraspect, the cellulosic material is corn cob. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material iswheat straw. In another aspect, the cellulosic material is switch grass.In another aspect, the cellulosic material is miscanthus. In anotheraspect, the cellulosic material is bagasse.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art. For example,physical pretreatment techniques can include various types of milling,irradiation, steaming/steam explosion, and hydrothermolysis; chemicalpretreatment techniques can include dilute acid, alkaline, organicsolvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlledhydrothermolysis; and biological pretreatment techniques can involveapplying lignin-solubilizing microorganisms (see, for example, Hsu,T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh, P., and Singh, A., 1993,Physicochemical and biological treatments for enzymatic/microbialconversion of lignocellulosic biomass, Adv. Appl. Microbiol. 39:295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: areview, in Enzymatic Conversion of Biomass for Fuels Production, Himmel,M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566,American Chemical Society, Washington, D.C., chapter 15; Gong, C. S.,Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production fromrenewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Olsson, L., and Hahn-Hagerdal, B.,1996, Fermentation of lignocellulosic hydrolysates for ethanolproduction, Enz. Microb. Tech. 18: 312-331; and Vallander, L., andEriksson, K.-E. L., 1990, Production of ethanol from lignocellulosicmaterials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” isdefined herein as a cellulosic material derived from corn stover bytreatment with heat and dilute acid.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide that is isolated from a source. In a preferredaspect, the polypeptide is at least 1% pure, preferably at least 5%pure, more preferably at least 10% pure, more preferably at least 20%pure, more preferably at least 40% pure, more preferably at least 60%pure, even more preferably at least 80% pure, and most preferably atleast 90% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 97%pure, more preferably at least 98% pure, even more preferably at least99% pure, most preferably at least 99.5% pure, and even most preferably100% pure by weight of the total polypeptide material present in thepreparation. The polypeptides of the present invention are preferably ina substantially pure form, i.e., that the polypeptide preparation isessentially free of other polypeptide material with which it is nativelyor recombinantly associated. This can be accomplished, for example, bypreparing the polypeptide by well-known recombinant methods or byclassical purification methods.

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having endoglucanase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having endoglucanase activity.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. The optional parameters usedare gap open penalty of 10, gap extension penalty of 0.5, and theEDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The outputof Needle labeled “longest identity” (obtained using the -nobriefoption) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein asa predicted protein that gives an E value (or expectancy score) of lessthan 0.001 in a tfasty search (Pearson, W. R., 1999, in BioinformaticsMethods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219)with the partial amino acid sequence of SEQ ID NO: 2 or the partialamino acid sequence SEQ ID NO: 4.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more (several) amino acids deleted fromthe amino and/or carboxyl terminus of an amino acid sequence comprisingthe partial amino acid sequence of SEQ ID NO: 2 or the partial aminoacid sequence of SEQ ID NO: 4; or a homologous sequence thereof; whereinthe fragment has endoglucanase activity.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more (several) nucleotides deleted from the 5′and/or 3′ end of a nucleotide sequence comprising the partial nucleotidesequence of SEQ ID NO: 1 or the partial nucleotide sequence of SEQ IDNO: 3; or a homologous sequence thereof; wherein the subsequence encodesa polypeptide fragment having endoglucanase activity.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide that is isolated from a source. In apreferred aspect, the polynucleotide is at least 1% pure, preferably atleast 5% pure, more preferably at least 10% pure, more preferably atleast 20% pure, more preferably at least 40% pure, more preferably atleast 60% pure, even more preferably at least 80% pure, and mostpreferably at least 90% pure, as determined by agarose electrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99% pure, and even most preferably at least99.5% pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form, i.e., that the polynucleotidepreparation is essentially free of other polynucleotide material withwhich it is natively or recombinantly associated. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, synthetic, or recombinant nucleotide sequence.

cDNA: The term “cDNA” is defined herein as a DNA molecule that can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that may bepresent in the corresponding genomic DNA. The initial, primary RNAtranscript is a precursor to mRNA that is processed through a series ofsteps before appearing as mature spliced mRNA. These steps include theremoval of intron sequences by a process called splicing. cDNA derivedfrom mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature or which is synthetic. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains the control sequences required forexpression of a coding sequence of the present invention.

Control sequences: The term “control sequences” is defined herein toinclude all components necessary for the expression of a polynucleotideencoding a polypeptide of the present invention. Each control sequencemay be native or foreign to the nucleotide sequence encoding thepolypeptide or native or foreign to each other. Such control sequencesinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, promoter, signal peptide sequence, andtranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Thecontrol sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleotide sequenceencoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of a polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the present invention and is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typethat is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of a polypeptide comprising an amino acid sequencecomprising the partial amino acid sequence of SEQ ID NO: 2 or thepartial amino acid sequence of SEQ ID NO: 4; or a homologous sequencethereof; as well as genetic manipulation of the DNA encoding such apolypeptide. The modification can be a substitution, a deletion and/oran insertion of one or more (several) amino acids as well asreplacements of one or more (several) amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having endoglucanase activity produced by anorganism expressing a modified polynucleotide sequence comprising anucleotide sequence comprising the partial nucleotide sequence of SEQ IDNO: 1 or the partial nucleotide sequence of SEQ ID NO: 3; or ahomologous sequence thereof. The modified nucleotide sequence isobtained through human intervention by modification of thepolynucleotide sequence comprising the partial nucleotide sequence ofSEQ ID NO: 1 or the partial nucleotide sequence of SEQ ID NO: 3; or ahomologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having EndoglucanaseActivity

In a first aspect, the present invention relates to isolatedpolypeptides comprising amino acid sequences comprising partial aminoacid sequences having a degree of identity to the partial amino acidsequence of SEQ ID NO: 2 or the partial amino acid sequence of SEQ IDNO: 4 of preferably at least 60%, more preferably at least 65%, morepreferably at least 70%, more preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 96%, at least 97%, at least 98%, or at least 99%, which haveendoglucanase activity (hereinafter “homologous polypeptides”). In apreferred aspect, the homologous polypeptides comprise amino acidsequences comprising partial amino acid sequences that differ by tenamino acids, preferably by five amino acids, more preferably by fouramino acids, even more preferably by three amino acids, most preferablyby two amino acids, and even most preferably by one amino acid from thepartial amino acid sequence of SEQ ID NO: 2 or the partial amino acidsequence of SEQ ID NO: 4.

In a preferred aspect, the polypeptide comprises an amino acid sequencecomprising the partial amino acid sequence of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof having endoglucanaseactivity. In another preferred aspect, the polypeptide comprises anamino acid sequence comprising the partial amino acid sequence of SEQ IDNO: 2.

In another preferred aspect, the polypeptide comprises an amino acidsequence comprising the partial amino acid sequence of SEQ ID NO: 4, oran allelic variant thereof; or a fragment thereof having endoglucanaseactivity. In another preferred aspect, the polypeptide comprises anamino acid sequence comprising the partial amino acid sequence of SEQ IDNO: 4.

In a second aspect, the present invention relates to isolatedpolypeptides having endoglucanase activity that are encoded bypolynucleotides comprising nucleotide sequences comprising partialnucleotide sequences that hybridize under preferably very low stringencyconditions, more preferably low stringency conditions, more preferablymedium stringency conditions, more preferably medium-high stringencyconditions, even more preferably high stringency conditions, and mostpreferably very high stringency conditions with (i) the partialnucleotide sequence of SEQ ID NO: 1 or the partial nucleotide sequenceof SEQ ID NO: 3, (ii) the cDNA sequence contained in the partialnucleotide sequence of SEQ ID NO: 1 or the partial nucleotide sequenceof SEQ ID NO: 3, (iii) a subsequence of (i) or (ii), or (iv) afull-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, New York). A subsequence of thepartial nucleotide sequence of SEQ ID NO: 1 contains at least 100contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmenthaving endoglucanase activity. In a preferred aspect, the complementarystrand is the full-length complementary strand of the partial nucleotidesequence of SEQ ID NO: 1 or the partial nucleotide sequence of SEQ IDNO: 3.

The nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or asubsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2or SEQ ID NO: 4; or a fragment thereof; may be used to design nucleicacid probes to identify and clone DNA encoding polypeptides havingendoglucanase activity from strains of different genera or speciesaccording to methods well known in the art. In particular, such probescan be used for hybridization with the genomic or cDNA of the genus orspecies of interest, following standard Southern blotting procedures, inorder to identify and isolate the corresponding gene therein. Suchprobes can be considerably shorter than the entire sequence, but shouldbe at least 14, preferably at least 25, more preferably at least 35, andmost preferably at least 70 nucleotides in length. It is, however,preferred that the nucleic acid probe is at least 100 nucleotides inlength. For example, the nucleic acid probe may be at least 200nucleotides in length. Both DNA and RNA probes can be used. The probesare typically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed bythe present invention.

A genomic DNA or cDNA library prepared from such other strains may,therefore, be screened for DNA that hybridizes with the probes describedabove and encodes a polypeptide having endoglucanase activity. Genomicor other DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that is homologous with SEQ ID NO: 1 orSEQ ID NO: 3; or a subsequence thereof; the carrier material ispreferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the partial nucleotide sequence of SEQ ID NO: 1 or thepartial nucleotide sequence of SEQ ID NO: 3; the cDNA sequence containedin the partial nucleotide sequence of SEQ ID NO: 1 or the partialnucleotide sequence of SEQ ID NO: 3; its full-length complementarystrand; or a subsequence thereof; under very low to very high stringencyconditions. Molecules to which the nucleic acid probe hybridizes underthese conditions can be detected using, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the partial nucleotidesequence of SEQ ID NO: 1 or a subsequence thereof. In another preferredaspect, the nucleic acid probe is the partial nucleotide sequence of SEQID NO: 1. In another preferred aspect, the nucleic acid probe is anucleotide sequence that encodes the partial amino acid sequence of SEQID NO: 2 or a subsequence thereof. In another preferred aspect, thenucleic acid probe is a nucleotide sequence that encodes the partialamino acid sequence of SEQ ID NO: 2.

In another preferred aspect, the nucleic acid probe is the partialnucleotide sequence of SEQ ID NO: 3 or a subsequence thereof. In anotherpreferred aspect, the nucleic acid probe is the partial nucleotidesequence of SEQ ID NO: 3. In another preferred aspect, the nucleic acidprobe is a nucleotide sequence that encodes the partial amino acidsequence of SEQ ID NO: 4 or a subsequence thereof. In another preferredaspect, the nucleic acid probe is a nucleotide sequence that encodes thepartial amino acid sequence of SEQ ID NO: 4.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at 45° C. (very low stringency), more preferably at50° C. (low stringency), more preferably at 55° C. (medium stringency),more preferably at 60° C. (medium-high stringency), even more preferablyat 65° C. (high stringency), and most preferably at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes of about 15 nucleotides to about 70 nucleotides inlength, the carrier material is washed once in 6×SCC plus 0.1% SDS for15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C.below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having endoglucanase activity encoded by polynucleotidescomprising nucleotide sequences comprising partial nucleotide sequencesthat have a degree of identity to the partial nucleotide sequence of SEQID NO: 1 or the partial nucleotide sequence of SEQ ID NO: 3 ofpreferably at least 60%, more preferably at least 65%, more preferablyat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, even more preferably at least 90%,most preferably at least 95%, and even most preferably at least 96%, atleast 97%, at least 98%, or at least 99%, which encode a polypeptidehaving endoglucanase activity. See polynucleotide section herein.

In a fourth aspect, the present invention relates to artificial variantscomprising an amino acid sequence comprising a substitution, deletion,and/or insertion of one or more (or several) amino acids of the partialamino acid sequence of SEQ ID NO: 2 or the partial amino acid sequenceof SEQ ID NO: 4; or a homologous sequence thereof. Preferably, aminoacid changes are of a minor nature, that is conservative amino acidsubstitutions or insertions that do not significantly affect the foldingand/or activity of the protein; small deletions, typically of one toabout 30 amino acids; small amino- or carboxyl-terminal extensions, suchas an amino-terminal methionine residue; a small linker peptide of up toabout 20-25 residues; or a small extension that facilitates purificationby changing net charge or another function, such as a poly-histidinetract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,endoglucanase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides that arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the partial amino acid sequence of SEQ ID NO: 2 or thepartial amino acid sequence of SEQ ID NO: 4 is 10, preferably 9, morepreferably 8, more preferably 7, more preferably at most 6, morepreferably 5, more preferably 4, even more preferably 3, most preferably2, and even most preferably 1.

Sources of Polypeptides Having Endoglucanase Activity

A polypeptide of the present invention may be obtained frommicroorganisms of any genus. For purposes of the present invention, theterm “obtained from” as used herein in connection with a given sourceshall mean that the polypeptide encoded by a nucleotide sequence isproduced by the source or by a strain in which the nucleotide sequencefrom the source has been inserted. In a preferred aspect, thepolypeptide obtained from a given source is secreted extracellularly.

A polypeptide having endoglucanase activity of the present invention maybe a bacterial polypeptide. For example, the polypeptide may be a grampositive bacterial polypeptide such as a Bacillus, Streptococcus,Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus,Clostridium, Geobacillus, or Oceanobacillus polypeptide havingendoglucanase activity, or a Gram negative bacterial polypeptide such asan E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasmapolypeptide having endoglucanase activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having endoglucanase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide having endoglucanaseactivity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingendoglucanase activity.

A polypeptide having endoglucanase activity of the present invention mayalso be a fungal polypeptide, and more preferably a yeast polypeptidesuch as a Candida, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia polypeptide having endoglucanaseactivity; or more preferably a filamentous fungal polypeptide such as anAcremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium,Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps,Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria,Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete,Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor,Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia,Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria polypeptide having endoglucanase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide having endoglucanaseactivity.

In another preferred aspect, the polypeptide is an Acremoniumcellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillusfumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillusnidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum,Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium zonatum, Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicolainsolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptidehaving endoglucanase activity.

In another preferred aspect, the polypeptide is a Penicilliumbrasilianum, Penicillium camembertii, Penicillium capsulatum,Penicillium chrysogenum, Penicillium citreonigrum, Penicillium citrinum,Peniciffiumclaviforme, Penicillium corylophilum, Penicillium crustosum,Penicillium digitatum, Penicillium expansum, Penicillium funiculosum,Penicillium glabrum, Penicillium granulatum, Penicillium griseofulvum,Penicillium islandicum, Penicillium italicum, Penicillium janthinellum,Penicillium lividum, Penicillium megasporum, Penicillium melinfi,Penicillium notatum, Penicillium oxalicum, Penicillium puberulum,Penicillium purpurescens, Penicillium purpurogenum, Penicilliumroquefortii, Penicillium rugulosum, Penicillium spinulosum, Penicilliumwaksmanii, or Penicillium sp. polypeptide having endoglucanase activity.

In a more preferred aspect, the polypeptide is a Penicillium brasilianumpolypeptide having endoglucanase activity, and most preferably aPenicillium brasilianum IBT 20888 polypeptide having endoglucanaseactivity.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques thatare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Uponsecretion of the fusion protein, the site is cleaved releasing thepolypeptide having endoglucanase activity from the fusion protein.Examples of cleavage sites include, but are not limited to, a Kex2 sitethat encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind.Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; andContreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising nucleotide sequences that encode polypeptides havingendoglucanase activity of the present invention.

In a preferred aspect, the polynucleotide comprises a nucleotidesequence comprising the partial nucleotide sequence of SEQ ID NO: 1 or asubsequence thereof that encodes a polypeptide fragment havingendoglucanase activity. In another preferred aspect, the polynucleotidecomprises a nucleotide sequence comprising the partial nucleotidesequence of SEQ ID NO: 1. The present invention also encompassesnucleotide sequences that encode polypeptides comprising amino acidsequences comprising the partial amino acid sequence of SEQ ID NO: 2,which differ from the partial nucleotide sequence of SEQ ID NO: 1 byvirtue of the degeneracy of the genetic code.

In another preferred aspect, the polynucleotide comprises a nucleotidesequence comprising the partial nucleotide sequence of SEQ ID NO: 3 or asubsequence thereof that encodes a polypeptide fragment havingendoglucanase activity. In another preferred aspect, the polynucleotidecomprises a nucleotide sequence comprising the partial nucleotidesequence of SEQ ID NO: 3. The present invention also encompassesnucleotide sequences that encode polypeptides comprising amino acidsequences comprising the partial amino acid sequence of SEQ ID NO: 4,which differ from the partial nucleotide sequence of SEQ ID NO: 3 byvirtue of the degeneracy of the genetic code.

The present invention also relates to mutant polynucleotides comprisinga nucleotide sequence comprising a partial nucleotide sequencecomprising at least one mutation in the partial nucleotide sequence ofSEQ ID NO: 1 or the partial nucleotide sequence of SEQ ID NO: 3, inwhich the mutant nucleotide sequence encodes a polypeptide comprising anamino acid sequence comprising the partial amino acid sequence of SEQ IDNO: 2 or the partial amino acid sequence of SEQ ID NO: 4, respectively.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Penicillium, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising nucleotide sequences comprising partial nucleotide sequenceshaving a degree of identity to the partial nucleotide sequence of SEQ IDNO: 1 or the partial nucleotide sequence of SEQ ID NO: 3 of preferablyat least 60%, more preferably at least 65%, more preferably at least70%, more preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, mostpreferably at least 95%, and even most preferably at least 96%, at least97%, at least 98%, or at least 99% identity, which encode a polypeptidehaving endoglucanase activity.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of anucleotide sequence comprising the partial nucleotide sequence of SEQ IDNO: 1 or the partial nucleotide sequence of SEQ ID NO: 3, e.g., asubsequence thereof, and/or by introduction of nucleotide substitutionsthat do not give rise to another amino acid sequence of the polypeptideencoded by the nucleotide sequence, but which correspond to the codonusage of the host organism intended for production of the enzyme, or byintroduction of nucleotide substitutions that may give rise to adifferent amino acid sequence. For a general description of nucleotidesubstitution, see, e.g., Ford et al., 1991, Protein Expression andPurification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide.

Amino acid residues essential to the activity of the polypeptide encodedby an isolated polynucleotide of the invention, and therefore preferablynot subject to substitution, may be identified according to proceduresknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (see, e.g., Cunningham and Wells, 1989, supra). In thelatter technique, mutations are introduced at every positively chargedresidue in the molecule, and the resultant mutant molecules are testedfor endoglucanase activity to identify amino acid residues that arecritical to the activity of the molecule. Sites of substrate-enzymeinteraction can also be determined by analysis of the three-dimensionalstructure as determined by such techniques as nuclear magnetic resonanceanalysis, crystallography or photoaffinity labeling (see, e.g., de Voset al., 1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992,supra).

The present invention also relates to isolated polynucleotides, encodingpolypeptides of the present invention, comprising nucleotide sequencescomprising partial nucleotide sequences that hybridize under very lowstringency conditions, preferably low stringency conditions, morepreferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with (i) the partialnucleotide sequence of SEQ ID NO: 1 or the partial nucleotide sequenceof SEQ ID NO: 3, (ii) the cDNA sequence contained in the partialnucleotide sequence of SEQ ID NO: 1 or the partial nucleotide sequenceof SEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or(ii); or allelic variants and subsequences thereof (Sambrook et al.,1989, supra), as defined herein. In a preferred aspect, thecomplementary strand comprises the full-length complementary strand ofthe partial nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

The present invention also relates to an isolated polynucleotidecomprising a nucleotide sequence comprising a partial nucleotidesequence obtained by (a) hybridizing a population of DNA under very low,low, medium, medium-high, high, or very high stringency conditions with(i) the partial nucleotide sequence of SEQ ID NO: 1 or the partialnucleotide sequence of SEQ ID NO: 3, (ii) the cDNA sequence contained inthe partial nucleotide sequence of SEQ ID NO: 1 or the partialnucleotide sequence of SEQ ID NO: 3, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (b) isolating the hybridizingpolynucleotide, which encodes a polypeptide having endoglucanaseactivity.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more (several) control sequences that direct the expression ofthe coding sequence in a suitable host cell under conditions compatiblewith the control sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence that is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences thatmediate the expression of the polypeptide. The promoter may be anynucleotide sequence that shows transcriptional activity in the host cellof choice including mutant, truncated, and hybrid promoters, and may beobtained from genes encoding extracellular or intracellular polypeptideseither homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator that is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding sequence thatencodes a signal peptide linked to the amino terminus of a polypeptideand directs the encoded polypeptide into the cell's secretory pathway.The 5′ end of the coding sequence of the nucleotide sequence mayinherently contain a signal peptide coding sequence naturally linked intranslation reading frame with the segment of the coding sequence thatencodes the secreted polypeptide. Alternatively, the 5′ end of thecoding sequence may contain a signal peptide coding sequence that isforeign to the coding sequence. The foreign signal peptide codingsequence may be required where the coding sequence does not naturallycontain a signal peptide coding sequence. Alternatively, the foreignsignal peptide coding sequence may simply replace the natural signalpeptide coding sequence in order to enhance secretion of thepolypeptide. However, any signal peptide coding sequence that directsthe expressed polypeptide into the secretory pathway of a host cell ofchoice, i.e., secreted into a culture medium, may be used in the presentinvention.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus stearothermophilusalpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the amino terminus of a polypeptide.The resultant polypeptide is known as a proenzyme or propolypeptide (ora zymogen in some cases). A propeptide is generally inactive and can beconverted to a mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei asparticproteinase, and Myceliophthora thermophila laccase (WO 95/33836).

Where both signal peptide and propeptide sequences are present at theamino terminus of a polypeptide, the propeptide sequence is positionednext to the amino terminus of a polypeptide and the signal peptidesequence is positioned next to the amino terminus of the propeptidesequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the nucleotide sequence encoding thepolypeptide would be operably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector that may include one or more(several) convenient restriction sites to allow for insertion orsubstitution of the nucleotide sequence encoding the polypeptide at suchsites. Alternatively, a polynucleotide sequence of the present inventionmay be expressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vectors of the present invention preferably contain one or more(several) selectable markers that permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers that confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity to the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMRβ1permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of the gene product. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention, which areadvantageously used in the recombinant production of the polypeptideshaving endoglucanase activity. A vector comprising a polynucleotide ofthe present invention is introduced into a host cell so that the vectoris maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive bacterium or a Gramnegative bacterium. Gram positive bacteria include, but not limited to,Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus,Lactobacillus, Lactococcus, Clostridium, Geobacillus, andOceanobacillus. Gram negative bacteria include, but not limited to, E.coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter,Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may also be any Streptococcus cell.Streptococcus cells useful in the practice of the present inventioninclude, but are not limited to, Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equisubsp. Zooepidemicus cells.

In a preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may also be any Streptomyces cell. Streptomycescells useful in the practice of the present invention include, but arenot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

In a preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-207, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art for introducing DNA into a host cell can be used.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium,Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix,Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia,Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus,Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium tropicum, Chrysosporium merdarium,Chrysosporium inops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Penicillium. In a morepreferred aspect, the cell is Penicillium brasilianum. In a mostpreferred aspect, the cell is Penicillium brasilianum IBT 20888.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell, as described herein, under conditions conducive for production ofthe polypeptide; and (b) recovering the polypeptide.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant polynucleotide comprising anucleotide sequence comprising at least one mutation in the partialnucleotide sequence of SEQ ID NO: 1 or the partial nucleotide sequenceof SEQ ID NO: 3, wherein the mutant polynucleotide encodes a polypeptidecomprising an amino acid sequence comprising the partial amino acidsequence of SEQ ID NO: 2 or the partial amino acid sequence of SEQ IDNO: 4; and (b) recovering the polypeptide.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotideencoding a polypeptide having endoglucanase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more (several) expression constructs encoding apolypeptide of the present invention into the plant host genome orchloroplast genome and propagating the resulting modified plant or plantcell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron that is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptidehaving endoglucanase activity of the present invention under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

Removal or Reduction of Endoglucanase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or more(several) nucleotides in the gene or a regulatory element required forthe transcription or translation thereof. For example, nucleotides maybe inserted or removed so as to result in the introduction of a stopcodon, the removal of the start codon, or a change in the open readingframe. Such modification or inactivation may be accomplished bysite-directed mutagenesis or PCR generated mutagenesis in accordancewith methods known in the art. Although, in principle, the modificationmay be performed in vivo, i.e., directly on the cell expressing thenucleotide sequence to be modified, it is preferred that themodification be performed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence that is then transformed into the parentcell to produce a defective gene. By homologous recombination, thedefective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of native and/or heterologouspolypeptides. Therefore, the present invention further relates tomethods of producing a native or heterologous polypeptide, comprising:(a) cultivating the mutant cell under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide. Theterm “heterologous polypeptides” is defined herein as polypeptides thatare not native to the host cell, a native protein in which modificationshave been made to alter the native sequence, or a native protein whoseexpression is quantitatively altered as a result of a manipulation ofthe host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of endoglucanase activityby fermentation of a cell that produces both a polypeptide of thepresent invention as well as the protein product of interest by addingan effective amount of an agent capable of inhibiting endoglucanaseactivity to the fermentation broth before, during, or after thefermentation has been completed, recovering the product of interest fromthe fermentation broth, and optionally subjecting the recovered productto further purification.

In a further aspect, the present invention relates to a method ofproducing a protein product essentially free of endoglucanase activityby cultivating the cell under conditions permitting the expression ofthe product, subjecting the resultant culture broth to a combined pH andtemperature treatment so as to reduce the endoglucanase activitysubstantially, and recovering the product from the culture broth.Alternatively, the combined pH and temperature treatment may beperformed on an enzyme preparation recovered from the culture broth. Thecombined pH and temperature treatment may optionally be used incombination with a treatment with an endoglucanase inhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the endoglucanase activity. Complete removal of endoglucanaseactivity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 2-4 or 9-11 and a temperature in the range of atleast 60-70° C. for a sufficient period of time to attain the desiredeffect, where typically, 30 to 60 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyendoglucanase-free product is of particular interest in the productionof eukaryotic polypeptides, in particular fungal proteins such asenzymes. The enzyme may be selected from, e.g., an amylolytic enzyme,lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase,chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, galactosidase,beta-galactosidase, glucoamylase, glucose oxidase, glucosidase,haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase,lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase,phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transferase, transglutaminase, or xylanase. Theendoglucanase-deficient cells may also be used to express heterologousproteins of pharmaceutical interest such as hormones, growth factors,receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from endoglucanase activity that is produced by amethod of the present invention.

Methods of Inhibiting Expression of a Polypeptide Having EndoglucanaseActivity

The present invention also relates to methods of inhibiting theexpression of a polypeptide having endoglucanase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA(siRNAs) for inhibiting transcription. In another preferred aspect, thedsRNA is micro RNA (miRNAs) for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the partial nucleotide sequence ofSEQ ID NO: 1 or the partial nucleotide sequence of SEQ ID NO: 3 forinhibiting expression of a polypeptide in a cell. While the presentinvention is not limited by any particular mechanism of action, thedsRNA can enter a cell and cause the degradation of a single-strandedRNA (ssRNA) of similar or identical sequences, including endogenousmRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencingtherapeutics. In one aspect, the invention provides methods toselectively degrade RNA using the dsRNA is of the present invention. Theprocess may be practiced in vitro, ex vivo or in vivo. In one aspect,the dsRNA molecules can be used to generate a loss-of-function mutationin a cell, an organ or an animal. Methods for making and using dsRNAmolecules to selectively degrade RNA are well known in the art, see, forexample, U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No.6,515,109; and U.S. Pat. No. 6,489,127.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that theendoglucanase activity of the composition has been increased, e.g., withan enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Processing of Cellulosic Material

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with a cellulolytic enzyme composition in the presence of apolypeptide having endoglucanase activity of the present invention. In apreferred aspect, the method further comprises recovering the degradedor converted cellulosic material.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with a cellulolytic enzyme composition in the presence of apolypeptide having endoglucanase activity of the present invention; (b)fermenting the saccharified cellulosic material of step (a) with one ormore fermenting microorganisms to produce the fermentation product; and(c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore fermenting microorganisms, wherein the cellulosic material issaccharified with a cellulolytic enzyme composition in the presence of apolypeptide having endoglucanase activity of the present invention andthe presence of the polypeptide having endoglucanase activity increasesthe degradation of the cellulosic material compared to the absence ofthe polypeptide having endoglucanase activity. In a preferred aspect,the fermenting of the cellulosic material produces a fermentationproduct. In another preferred aspect, the method further comprisesrecovering the fermentation product from the fermentation.

The composition and the polypeptide having endoglucanase activity can bein the form of a crude fermentation broth with or without the cellsremoved or in the form of a semi-purified or purified enzyme preparationor the composition can comprise a host cell of the present invention asa source of the polypeptide having endoglucanase activity in afermentation process with the biomass.

The methods of the present invention can be used to saccharify acellulosic material to fermentable sugars and convert the fermentablesugars to many useful substances, e.g., chemicals and fuels. Theproduction of a desired fermentation product from cellulosic materialtypically involves pretreatment, enzymatic hydrolysis(saccharification), and fermentation.

The processing of cellulosic material according to the present inventioncan be accomplished using processes conventional in the art. Moreover,the methods of the present invention can be implemented using anyconventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and cofermentation (SSCF); hybridhydrolysis and fermentation (HHF); SHCF (separate hydrolysis andco-fermentation), HHCF (hybrid hydrolysis and fermentation), and directmicrobial conversion (DMC). SHF uses separate process steps to firstenzymatically hydrolyze lignocellulose to fermentable sugars, e.g.,glucose, cellobiose, cellotriose, and pentose sugars, and then fermentthe fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis oflignocellulose and the fermentation of sugars to ethanol are combined inone step (Philippidis, G. P., 1996, Cellulose bioconversion technology,in Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,ed., Taylor & Francis, Washington, D.C., 179-212). SSCF involves thecofermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999,Enzymes, energy and the environment: A strategic perspective on the U.S.Department of Energy's research and development activities forbioethanol, Biotechnol. Prog. 15: 817-827). HHF involves a separatehydrolysis separate step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, lignocellulose hydrolysis, and fermentation) in oneor more steps where the same organism is used to produce the enzymes forconversion of the lignocellulose to fermentable sugars and to convertthe fermentable sugars into a final product (Lynd, L. R., Weimer, P. J.,van Zyl, W. H., and Pretorius, I. S., 2002, Microbial celluloseutilization: Fundamentals and biotechnology, Microbiol. Mol. Biol.Reviews 66: 506-577). It is understood herein that any method known inthe art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Fernandade Castilhos Corazza, Flávio Faria de Moraes, Gisella Maria Zanin andIvo Neitzel, 2003, Optimal control in fed-batch reactor for thecellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov,A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysisof cellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee,J. M., 1983, Bioconversion of waste cellulose by using an attritionbioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensivestirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn,A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., 1996,Enhancement of enzymatic cellulose hydrolysis using a novel type ofbioreactor with intensive stirring induced by electromagnetic field,Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor typesinclude: Fluidized bed, upflow blanket, immobilized, and extruder typereactors for hydrolysis and/or fermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents. The cellulosic material can also be subjected topre-soaking, wetting, or conditioning prior to pretreatment usingmethods known in the art. Conventional pretreatments include, but arenot limited to, steam pretreatment (with or without explosion), diluteacid pretreatment, hot water pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ultrasound, electroporation, microwave, supercritical CO₂,supercritical H₂O, and ammonia percolation.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with hydrolysis, such as simultaneously with treatment ofthe cellulosic material with one or more cellulolytic enzymes, or otherenzyme activities, to release fermentable sugars, such as glucose and/ormaltose. In most cases the pretreatment step itself results in someconversion of biomass to fermentable sugars (even in absence ofenzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulase, accessible to enzymes. The lignocellulose materialis passed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably done at 140-230° C., more preferably 160-200° C., and mostpreferably 170-190° C., where the optimal temperature range depends onany addition of a chemical catalyst. Residence time for the steampretreatment is preferably 1-15 minutes, more preferably 3-12 minutes,and most preferably 4-10 minutes, where the optimal residence timedepends on temperature range and any addition of a chemical catalyst.Steam pretreatment allows for relatively high solids loadings, so thatthe cellulosic material is generally only moist during the pretreatment.The steam pretreatment is often combined with an explosive discharge ofthe material after the pretreatment, which is known as steam explosion,that is, rapid flashing to atmospheric pressure and turbulent flow ofthe material to increase the accessible surface area by fragmentation(Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe andZacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. PatentApplication No. 20020164730). During steam pretreatment, hemicelluloseacetyl groups are cleaved and the resulting acid autocatalyzes partialhydrolysis of the hemicellulose to monosaccharides and oligosaccharides.Lignin is removed to only a limited extent.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 3% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762).

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Examples of suitable chemicalpretreatment processes include, for example, dilute acid pretreatment,lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX),ammonia percolation (APR), and organosolv pretreatments.

In dilute acid pretreatment, the cellulosic material is mixed withdilute acid, typically H₂SO₄, and water to form a slurry, heated bysteam to the desired temperature, and after a residence time flashed toatmospheric pressure. The dilute acid pretreatment can be performed witha number of reactor designs, e.g., plug-flow reactors, counter-currentreactors, or continuous counter-current shrinking bed reactors (Duff andMurray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to, limepretreatment, wet oxidation, ammonia percolation (APR), and ammoniafiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium carbonate, sodium hydroxide,or ammonia at low temperatures of 85-150° C. and residence times from 1hour to several days (Wyman et al., 2005, Bioresource Technol. 96:1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclosepretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed at preferably 1-40% drymatter, more preferably 2-30% dry matter, and most preferably 5-20% drymatter, and often the initial pH is increased by the addition of alkalisuch as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion), can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating cellulosic materialwith liquid or gaseous ammonia at moderate temperatures, such as 90-100°C., and high pressure, such as 17-20 bar, for 5-10 minutes, where thedry matter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121:1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). AFEX pretreatment results in the depolymerization ofcellulose and partial hydrolysis of hemicellulose. Lignin-carbohydratecomplexes are cleaved.

Organosolv pretreatment delignifies cellulosic material by extractionusing aqueous ethanol (40-60% ethanol) at 160-200° C. for 30-60 minutes(Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006,Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem.Biotechnol. 121:219-230). Sulphuric acid is usually added as a catalyst.In organosolv pretreatment, the majority of the hemicellulose isremoved.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as anacid treatment, and more preferably as a continuous dilute and/or mildacid treatment. The acid is typically sulfuric acid, but other acids canalso be used, such as acetic acid, citric acid, nitric acid, phosphoricacid, tartaric acid, succinic acid, hydrogen chloride, or mixturesthereof. Mild acid treatment is conducted in the pH range of preferably1-5, more preferably 1-4, and most preferably 1-3. The acidconcentration is in the range from preferably 0.01 to 20 wt % acid, morepreferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt %acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contactedwith the cellulosic material and held at a temperature in the range ofpreferably 160-220° C., and more preferably 165-195° C., for periodsranging from seconds to minutes to, e.g., 1 second to 60 minutes.

In another aspect, pretreatment is carried out as an ammonia fiberexplosion step (AFEX pretreatment step).

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, more preferablybetween 20-70 wt %, and most preferably between 30-60 wt %, such asaround 50 wt %. The pretreated cellulosic material can be unwashed orwashed using any method known in the art, e.g., washed with water.

Mechanical Pretreatment: The term “mechanical pretreatment” refers tovarious types of grinding or milling (e.g., dry milling, wet milling, orvibratory ball milling).

Physical Pretreatment: The term “physical pretreatment” refers to anypretreatment that promotes the separation and/or release of cellulose,hemicellulose, and/or lignin from cellulosic material. For example,physical pretreatment can involve irradiation (e.g., microwaveirradiation), steaming/steam explosion, hydrothermolysis, andcombinations thereof.

Physical pretreatment can involve high pressure and/or high temperature(steam explosion). In one aspect, high pressure means pressure in therange of preferably about 300 to about 600 psi, more preferably about350 to about 550 psi, and most preferably about 400 to about 500 psi,such as around 450 psi. In another aspect, high temperature meanstemperatures in the range of about 100 to about 300° C., preferablyabout 140 to about 235° C. In a preferred aspect, mechanicalpretreatment is performed in a batch-process, steam gun hydrolyzersystem that uses high pressure and high temperature as defined above,e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

Combined Physical and Chemical Pretreatment: The cellulosic material canbe pretreated both physically and chemically. For instance, thepretreatment step can involve dilute or mild acid treatment and hightemperature and/or pressure treatment. The physical and chemicalpretreatments can be carried out sequentially or simultaneously, asdesired. A mechanical pretreatment can also be included.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto mechanical, chemical, or physical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996,Pretreatment of biomass, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; Ghosh and Singh, 1993, Physicochemical and biologicaltreatments for enzymatic/microbial conversion of cellulosic biomass,Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreatinglignocellulosic biomass: a review, in Enzymatic Conversion of Biomassfor Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P.,eds., ACS Symposium Series 566, American Chemical Society, Washington,D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996,Fermentation of lignocellulosic hydrolysates for ethanol production,Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990,Production of ethanol from lignocellulosic materials: State of the art,Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the pretreatedcellulosic material is hydrolyzed to break down cellulose andalternatively also hemicellulose to fermentable sugars, such as glucose,xylose, xylulose, arabinose, maltose, mannose, galactose, or solubleoligosaccharides. The hydrolysis is performed enzymatically by acellulolytic enzyme composition in the presence of a polypeptide havingendoglucanase activity of the present invention. One or more of theenzymes of the composition can also be added sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In a preferred aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the pretreated cellulosic material (substrate)is fed gradually to, for example, an enzyme containing hydrolysissolution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 96 hours, more preferably about 16 to about 72 hours,and most preferably about 24 to about 48 hours. The temperature is inthe range of preferably about 25° C. to about 70° C., more preferablyabout 30° C. to about 65° C., and more preferably about 40° C. to 60°C., in particular about 50° C. The pH is in the range of preferablyabout 3 to about 8, more preferably about 3.5 to about 7, and mostpreferably about 4 to about 6, in particular about pH 5. The dry solidscontent is in the range of preferably about 5 to about 50 wt %, morepreferably about 10 to about 40 wt %, and most preferably about 20 toabout 30 wt %.

In addition to a polypeptide having endoglucanase activity of thepresent invention, the cellulolytic enzyme composition may comprise anyprotein involved in the processing of a cellulose-containing material toglucose, and/or hemicellulose to xylose, mannose, galactose, andarabinose, their polymers, or products derived from them as describedbelow. In one aspect, the cellulolytic enzyme composition comprises oneor more enzymes selected from the group consisting of a cellulase, anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the cellulolytic enzyme composition further or even furthercomprises a polypeptide having cellulolytic enhancing activity. See, forexample, WO 2005/074647, WO 2005/074656, and WO 2007/089290. In anotheraspect, the cellulolytic enzyme composition further or even furthercomprises one or more additional enzyme activities to improve thedegradation of the cellulose-containing material. Preferred additionalenzymes are hemicellulases, esterases (e.g., lipases, phospholipases,and/or cutinases), proteases, laccases, peroxidases, or mixturesthereof.

The enzymes can be derived or obtained from any suitable origin,including, bacterial, fungal, yeast, plant, or mammalian origin. Theterm “obtained” means herein that the enzyme may have been isolated froman organism that naturally produces the enzyme as a native enzyme. Theterm “obtained” also means herein that the enzyme may have been producedrecombinantly in a host organism employing methods described herein,wherein the recombinantly produced enzyme is either native or foreign tothe host organism or has a modified amino acid sequence, e.g., havingone or more amino acids that are deleted, inserted and/or substituted,i.e., a recombinantly produced enzyme that is a mutant and/or a fragmentof a native amino acid sequence or an enzyme produced by nucleic acidshuffling processes known in the art. Encompassed within the meaning ofa native enzyme are natural variants and within the meaning of a foreignenzyme are variants obtained recombinantly, such as by site-directedmutagenesis or shuffling.

The cellulolytic enzyme composition may be a monocomponent preparation,e.g., an endoglucanase, a multicomponent preparation, e.g.,endoglucanase(s), cellobiohydrolase(s), and beta-glucosidase(s), or acombination of multicomponent and monocomponent protein preparations.The cellulolytic proteins may have activity, i.e., hydrolyze thecellulose-containing material, either in the acid, neutral, or alkalinepH-range. One or more components of the cellulolytic enzyme compositionmay be a recombinant component, i.e., produced by cloning of a DNAsequence encoding the single component and subsequent cell transformedwith the DNA sequence and expressed in a host (see, for example, WO91/17243 and WO 91/17244). The host is preferably a heterologous host(enzyme is foreign to host), but the host may under certain conditionsalso be a homologous host (enzyme is native to host). Monocomponentcellulolytic proteins may also be prepared by purifying such a proteinfrom a fermentation broth.

The enzymes used in the present invention can be in any form suitablefor use in the methods described herein, such as a crude fermentationbroth with or without cells or substantially pure polypeptides. Theenzyme(s) can be a dry powder or granulate, a non-dusting granulate, aliquid, a stabilized liquid, or a protected enzyme(s). Liquid enzymepreparations can, for instance, be stabilized by adding stabilizers suchas a sugar, a sugar alcohol or another polyol, and/or lactic acid oranother organic acid according to established process. Protected enzymescan be prepared according to the process disclosed in EP 238,216.

Examples of commercial cellulolytic protein preparations suitable foruse in the present invention include, for example, CELLUCLAST™(available from Novozymes NS) and NOVOZYM™ 188 (available from NovozymesNS). Other commercially available preparations comprising cellulase thatmay be used include CELLUZYME™, CEREFLO™ and ULTRAFLO™ (Novozymes NS),LAMINEX™ and SPEZYME™ CP (Genencor Int.), ROHAMENT™ 7069 W (Röhm GmbH),and FIBREZYME® LDI, FIBREZYME® LBR, or VISCOSTAR® 150L (DyadicInternational, Inc., Jupiter, Fla., USA). The cellulase enzymes areadded in amounts effective from about 0.001% to about 5.0% wt. ofsolids, more preferably from about 0.025% to about 4.0% wt. of solids,and most preferably from about 0.005% to about 2.0% wt. of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the methods of thepresent invention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GENBANK™accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo,et al., 1988, Gene 63:11-22; GENBANK™ accession no. M19373); Trichodermareesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol.64: 555-563; GENBANK™ accession no. AB003694); Trichoderma reeseiendoglucanase IV (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591;GENBANK™ accession no. Y11113); and Trichoderma reesei endoglucanase V(Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GENBANK™accession no. Z33381); Aspergillus aculeatus endoglucanase (Ooi et al.,1990, Nucleic Acids Research 18: 5884); Aspergillus kawachiiendoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439);Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90:9-14); Fusarium oxysporum endoglucanase (GENBANK™ accession no. L29381);Humicola grisea var. thermoidea endoglucanase (GENBANK™ accession no.AB003107); Melanocarpus albomyces endoglucanase (GENBANK™ accession no.MAL515703); Neurospora crassa endoglucanase (GENBANK™ accession no.XM_(—)324477); Humicola insolens endoglucanase V; Myceliophthorathermophila CBS 117.65 endoglucanase; basidiomycete CBS 495.95endoglucanase; basidiomycete CBS 494.95 endoglucanase; Thielaviaterrestris NRRL 8126 CEL6B endoglucanase; Thielavia terrestris NRRL 8126CEL6C endoglucanase); Thielavia terrestris NRRL 8126 CEL7Cendoglucanase; Thielavia terrestris NRRL 8126 CEL7E endoglucanase;Thielavia terrestris NRRL 8126 CEL7F endoglucanase; Cladorrhinumfoecundissimum ATCC 62373 CEL7A endoglucanase; and Trichoderma reeseistrain No. VTT-D-80133 endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the methods of the presentinvention include, but are not limited to, Trichoderma reeseicellobiohydrolase I; Trichoderma reesei cellobiohydrolase II; Humicolainsolens cellobiohydrolase I, Myceliophthora thermophilacellobiohydrolase II, Thielavia terrestris cellobiohydrolase II (CEL6A),Chaetomium thermophilum cellobiohydrolase I, and Chaetomium thermophilumcellobiohydrolase II.

Examples of beta-glucosidases useful in the methods of the presentinvention include, but are not limited to, Aspergillus oryzaebeta-glucosidase; Aspergillus fumigatus beta-glucosidase; Penicilliumbrasilianum IBT 20888 beta-glucosidase; Aspergillus nigerbeta-glucosidase; and Aspergillus aculeatus beta-glucosidase.

The Aspergillus oryzae polypeptide having beta-glucosidase activity canbe obtained according to WO 2002/095014. The Aspergillus fumigatuspolypeptide having beta-glucosidase activity can be obtained accordingto WO 2005/047499. The Penicillium brasilianum polypeptide havingbeta-glucosidase activity can be obtained according to WO 2007/019442.The Aspergillus niger polypeptide having beta-glucosidase activity canbe obtained according to Dan et al., 2000, J. Biol. Chem. 275:4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidaseactivity can be obtained according to Kawaguchi et al., 1996, Gene 173:287-288.

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BGfusion protein or the Aspergillus oryzae beta-glucosidase fusion proteinobtained according to WO 2008/057637.

Other endoglucanases, cellobiohydrolases, and beta-glucosidases aredisclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat B., 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat.No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S.Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

In the methods of the present invention, any polypeptide havingcellulolytic enhancing activity can be used.

In a first aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motifs:

[ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)- [HNQ]and [FW]-[TF]-K-[AIV],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5contiguous positions, and X(4) is any amino acid at 4 contiguouspositions.

The polypeptide comprising the above-noted motifs may further comprise:

H-X(1,2)-G-P-X(3)-[YW]-[AILMV],[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], orH-X(1,2)-G-P-X(3)-[YW]-[AILMV] and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV],

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2contiguous positions, X(3) is any amino acid at 3 contiguous positions,and X(2) is any amino acid at 2 contiguous positions. In the abovemotifs, the accepted IUPAC single letter amino acid abbreviation isemployed.

In a preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In anotherpreferred aspect, the isolated polypeptide having cellulolytic enhancingactivity further comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. Inanother preferred aspect, the polypeptide having cellulolytic enhancingactivity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and[EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

In a second aspect, the polypeptide having cellulolytic enhancingactivity comprises the following motif:

[ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)- A-[HNQ],

wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5contiguous positions, and x(3) is any amino acid at 3 contiguouspositions. In the above motif, the accepted IUPAC single letter aminoacid abbreviation is employed.

Examples of polypeptides having cellulolytic enhancing activity usefulin the methods of the present invention include, but are not limited to,polypeptides having cellulolytic enhancing activity from Thielaviaterrestris (WO 2005/074647); polypeptides having cellulolytic enhancingactivity from Thermoascus aurantiacus (WO 2005/074656); and polypeptideshaving cellulolytic enhancing activity from Trichoderma reesei. (WO2007/089290).

The cellulolytic enzymes and proteins used in the methods of the presentinvention may be produced by fermentation of the above-noted microbialstrains on a nutrient medium containing suitable carbon and nitrogensources and inorganic salts, using procedures known in the art (see,e.g., Bennett, J. W. and LaSure, L. (eds.), More Gene Manipulations inFungi, Academic Press, CA, 1991). Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand cellulolytic enzyme production are known in the art (see, e.g.,Bailey, J. E., and Ollis, D. F., Biochemical Engineering Fundamentals,McGraw-Hill Book Company, NY, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of a cellulolytic enzyme. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe cellulolytic enzyme to be expressed or isolated. The resultingcellulolytic enzymes produced by the methods described above may berecovered from the fermentation medium and purified by conventionalprocedures.

The optimum amounts of the enzymes and polypeptides having endoglucanaseactivity depend on several factors including, but not limited to, themixture of component cellulolytic enzymes, the cellulosic substrate, theconcentration of cellulosic substrate, the pretreatment(s) of thecellulosic substrate, temperature, time, pH, and inclusion of fermentingorganism (e.g., yeast for Simultaneous Saccharification andFermentation).

In a preferred aspect, an effective amount of cellulolytic enzyme(s) tocellulosic material is about 0.5 to about 50 mg, preferably at about 0.5to about 40 mg, more preferably at about 0.5 to about 25 mg, morepreferably at about 0.75 to about 20 mg, more preferably at about 0.75to about 15 mg, even more preferably at about 0.5 to about 10 mg, andmost preferably at about 2.5 to about 10 mg per g of cellulosicmaterial.

In another preferred aspect, an effective amount of a polypeptide havingendoglucanase activity to cellulosic material is about 0.01 to about 50mg, preferably at about 0.5 to about 40 mg, more preferably at about 0.5to about 25 mg, more preferably at about 0.75 to about 20 mg, morepreferably at about 0.75 to about 15 mg, even more preferably at about0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg perg of cellulosic material.

In another preferred aspect, an effective amount of polypeptide(s)having endoglucanase activity to cellulolytic enzyme(s) is about 0.005to about 1.0 g, preferably at about 0.01 to about 1.0 g, more preferablyat about 0.15 to about 0.75 g, more preferably at about 0.15 to about0.5 g, more preferably at about 0.1 to about 0.5 g, even more preferablyat about 0.1 to about 0.5 g, and most preferably at about 0.05 to about0.2 g per g of cellulolytic enzyme(s).

Fermentation.

The fermentable sugars obtained from the pretreated and hydrolyzedcellulosic material can be fermented by one or more fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous. Such methods include, but are not limited to, separatehydrolysis and fermentation (SHF); simultaneous saccharification andfermentation (SSF); simultaneous saccharification and cofermentation(SSCF); hybrid hydrolysis and fermentation (HHF); SHCF (separatehydrolysis and co-fermentation), HHCF (hybrid hydrolysis andfermentation), and direct microbial conversion (DMC).

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art. Examples of substrates suitablefor use in the methods of present invention, include cellulosicmaterials, such as wood or plant residues or low molecular sugars DP1-3obtained from processed cellulosic material that can be metabolized bythe fermenting microorganism, and which can be supplied by directaddition to the fermentation medium.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be C₆ and/or C₅ fermenting organisms, or a combinationthereof. Both C₆ and C₅ fermenting organisms are well known in the art.Suitable fermenting microorganisms are able to ferment, i.e., convert,sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose,galactose, or oligosaccharides, directly or indirectly into the desiredfermentation product.

Examples of bacterial and fungal fermenting organisms producing ethanolare described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69:627-642.

Examples of fermenting microorganisms that can ferment C6 sugars includebacterial and fungal organisms, such as yeast. Preferred yeast includesstrains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.

Examples of fermenting organisms that can ferment C5 sugars includebacterial and fungal organisms, such as yeast. Preferred C5 fermentingyeast include strains of Pichia, preferably Pichia stipitis, such asPichia stipitis CBS 5773; strains of Candida, preferably Candidaboidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candidapseudotropicalis, or Candida utilis.

Other fermenting organisms include strains of Zymomonas, such asZymomonas mobilis; Hansenula, such as Hansenula anomala; Klyveromyces,such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli,especially E. coli strains that have been genetically modified toimprove the yield of ethanol.

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida. In another more preferredaspect, the yeast is Candida boidinii. In another more preferred aspect,the yeast is Candida brassicae. In another more preferred aspect, theyeast is Candida diddensii. In another more preferred aspect, the yeastis Candida pseudotropicalis. In another more preferred aspect, the yeastis Candida utilis. In another preferred aspect, the yeast is aClavispora. In another more preferred aspect, the yeast is Clavisporalusitaniae. In another more preferred aspect, the yeast is Clavisporaopuntiae. In another preferred aspect, the yeast is a Pachysolen. Inanother more preferred aspect, the yeast is Pachysolen tannophilus. Inanother preferred aspect, the yeast is a Pichia. In another morepreferred aspect, the yeast is a Pichia stipitis. In another preferredaspect, the yeast is a Bretannomyces. In another more preferred aspect,the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996,Cellulose bioconversion technology, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212).

Bacteria that can efficiently ferment hexose and pentose to ethanolinclude, for example, Zymomonas mobilis and Clostridium thermocellum(Philippidis, 1996, supra).

In a preferred aspect, the bacterium is a Zymomonas. In a more preferredaspect, the bacterium is Zymomonas mobilis. In another preferred aspect,the bacterium is a Clostridium. In another more preferred aspect, thebacterium is Clostridium thermocellum.

Commercially available yeast suitable for ethanol production includes,e.g., ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI™(available from Fleischmann's Yeast, USA), SUPERSTART™ and THERMOSACC™fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM™ AFTand XR (available from NABC—North American Bioproducts Corporation, GA,USA), GERT STRAND™ (available from Gert Strand AB, Sweden), and FERMIOL™(available from DSM Specialties).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning andimproving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470).

In a preferred aspect, the genetically modified fermenting microorganismis Saccharomyces cerevisiae. In another preferred aspect, thegenetically modified fermenting microorganism is Zymomonas mobilis. Inanother preferred aspect, the genetically modified fermentingmicroorganism is Escherichia coli. In another preferred aspect, thegenetically modified fermenting microorganism is Klebsiella oxytoca.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedlignocellulose or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, such as about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., inparticular about 32° C. or 50° C., and at about pH 3 to about pH 8, suchas around pH 4-5, 6, or 7.

In a preferred aspect, the yeast and/or another microorganism is appliedto the degraded lignocellulose or hydrolysate and the fermentation isperformed for about 12 to about 96 hours, such as typically 24-60 hours.In a preferred aspect, the temperature is preferably between about 20°C. to about 60° C., more preferably about 25° C. to about 50° C., andmost preferably about 32° C. to about 50° C., in particular about 32° C.or 50° C., and the pH is generally from about pH 3 to about pH 7,preferably around pH 4-7. However, some, e.g., bacterial fermentingorganisms have higher fermentation temperature optima. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 2×10⁸ viable cell count per ml of fermentation broth.Further guidance in respect of using yeast for fermentation can be foundin, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D.R. Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

For ethanol production, following the fermentation the fermented slurryis distilled to extract the ethanol. The ethanol obtained according tothe methods of the invention can be used as, e.g., fuel ethanol,drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator can be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenoreet al., Improving ethanol production and viability of Saccharomycescerevisiae by a vitamin feeding strategy during fed-batch process,Springer-Verlag (2002), which is hereby incorporated by reference.Examples of minerals include minerals and mineral salts that can supplynutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g., aceticacid, acetonic acid, adipic acid, ascorbic acid, citric acid,2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid,gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid,itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid,propionic acid, succinic acid, and xylonic acid); a ketone (e.g.,acetone); an amino acid (e.g., aspartic acid, glutamic acid, glycine,lysine, serine, and threonine); and a gas (e.g., methane, hydrogen (H₂),carbon dioxide (CO₂), and carbon monoxide (CO)). The fermentationproduct can also be protein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is arabinitol. In another more preferred aspect, the alcohol isbutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is glycerol. In another morepreferred aspect, the alcohol is methanol. In another more preferredaspect, the alcohol is 1,3-propanediol. In another more preferredaspect, the alcohol is sorbitol. In another more preferred aspect, thealcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M.M., and Jonas, R., 2002, The biotechnological production of sorbitol,Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D.,1995, Processes for fermentative production of xylitol—a sugarsubstitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi,N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanolby Clostridium beijerinckii BA101 and in situ recovery by gas stripping,World Journal of Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard, A., andMargaritis, A., 2004, Empirical modeling of batch fermentation kineticsfor poly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87 (4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies onhydrogen production by continuous culture system of hydrogen-producinganaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; andGunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114,1997, Anaerobic digestion of biomass for methane production: A review.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol.% can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

DNA Sequencing

DNA sequencing was performed using an Applied Biosystems Model 3130XGenetic Analyzer (Applied Biosystems, Foster City, Calif., USA) usingdye terminator chemistry (Giesecke et al., 1992, Journal of Virol.Methods 38: 47-60). Sequences were assembled using phred/phrap/consed(University of Washington, Seattle, Wash., USA) with sequence specificprimers.

Strains

Penicillium brasilianum strain IBT 20888 (IBT Culture Collection ofFungi, Technical University of Denmark, Copenhagen, Denmark) was used assource of the endoglucanase genes.

Media and Solutions

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of sodium chloride.

LB ampicillin medium was composed per liter of 10 g of tryptone, 5 g ofyeast extract, 5 g of sodium chloride, and 50 μg of ampicillin per ml(filter sterilized, added after autoclaving).

LB ampicillin plates were composed per liter of LB ampicillin medium and15 g of bacto agar.

TE was composed of 10 mM Tris pH 7.4 and 0.1 mM EDTA.

Example 1 Isolation of Genomic DNA from Penicillium brasilianum IBT20888

Spores of Penicillium brasilianum strain IBT 20888 were propagated onrice according to Carlsen, 1994, Ph.D. thesis, Department ofBiotechnology, The Technical University of Denmark. The spores wererecovered with 20 ml of 0.1% TWEEN® 20 and inoculated at a concentrationof 1×10⁶ spores per ml into 100 ml of Mandels and Weber medium (Mandelsand Weber, 1969, Adv. Chem. Ser. 95: 394-414) containing 1% glucosesupplemented per liter with 0.25 g of yeast extract and 0.75 g ofBactopeptone in a 500 ml baffled shake flask. The fungal mycelia wereharvested after 24 hours of aerobic growth at 30° C., 150 rpm.

Mycelia were collected by filtration through a NALGENE® DS0281-5000filter (Nalge Nunc International Corporation, Rochester, N.Y., USA)until dryness and frozen in liquid nitrogen. The frozen mycelia wereground to a powder in a dry ice chilled mortar and distributed to ascrew-cap tube. The powder was suspended in a total volume of 40 ml of50 mM CAPS (3-(cyclohexylamino)-1-propanesulfonic acid)-NaOH pH 11buffer containing 0.5% lithium dodecyl sulfate and 0.5 mM EDTA. Thesuspension was placed at 60° C. for 2 hours and periodically resuspendedby inversion. To the suspension was added an equal volume ofphenol:chloroform (1:1 v/v) neutralized with 0.1 M Tris base, and thetube was mixed on a rotating wheel at 37° C. for 2 hours. Aftercentrifugation at 2500 rpm for 10 minutes in a Sorvall H1000B rotor, theaqueous phase (top phase) was re-extracted again with phenol:chloroform(1:1 v/v) and centrifuged at 15,000×g for 5 minutes. The aqueous phasefrom the second extraction was brought to 2.5 M ammonium acetate (stock10 M) and placed at −20° C. until frozen. After thawing, the extract wascentrifuged at 15,000×g for 20 minutes in a cold rotor. The pellet(primarily rRNA) was discarded and the nucleic acids in the supernatantwere precipitated by addition of 0.7 volumes of isopropanol. Aftercentrifugation at 15,000×g for 15 minutes, the pellet was rinsed threetimes with 5 ml of 70% ethanol (without resuspension), air-dried almostcompletely, and dissolved in 1.0 ml of 0.1×TE. The dissolved pellet wastransferred to two 1.5 ml microfuges tubes. The nucleic acids wereprecipitated by addition of ammonium acetate (0.125 ml) to 2.0 M andethanol to 63% (1.07 ml) and centrifuged at maximum speed for 10 minutesin a Sorvall MC 12V microcentrifuge (Kendro Laboratory Products,Asheville, N.C., USA). The pellet was rinsed twice with 70% ethanol,air-dried completely, and dissolved in 500 μl of 0.1×TE.

Example 2 PCR Amplification of the Cel5a and Cel5b Endoglucanase Genesfrom Penicillium brasilianum IBT 20888

Based on database sequences of other Family 5 endoglucanases and on theN-terminal amino acid sequence of a purified Penicillium brasilianumendoglucanase CEL5C (WO 2007/109441), a forward primer was designedusing the CODEHOP strategy (Rose et al., 1998, Nucleic Acids Res. 26:1628-35). From database information on conserved regions in other Family5 endoglucanases, two reverse primers were designed as shown below usingthe CODEHOP strategy.

Forward primer, Fwd: (SEQ ID NO: 5) 5′-TTCGGTACCTCTGAGTCTGGNGCNGARTT-3′Reverse primer, Rev1: (SEQ ID NO: 6)5′-TGATCCATATCGTGGTACTCGTTRTTNGTRTCRAA-3′ Reverse primer, Rev2:(SEQ ID NO: 7) 5′-CCGTTGTAGCGACCGTARTTRTGNGGRTC-3′where R=A or G, Y═C or T, K=G or T and N=A, C, G or T

Amplification reactions (30 μl) were prepared using approximately 1 μgof Penicillium brasilianum genomic DNA as template. In addition, eachreaction contained 30 pmol of the forward primer, 30 pmol of the reverseprimer, 200 μM each of dATP, dCTP, dGTP, and dTTP, 1× AMPLITAQ® DNApolymerase buffer (Applied Biosystems, Foster City, Calif., USA), and0.5 unit of AMPLITAQ® DNA polymerase (5.0 U/μl, Applied Biosystems,Foster City, Calif., USA).

The reactions were incubated in a ROBOCYCLER® temperature cycler(Stratagene, La Jolla, Calif., USA) programmed for 1 cycle at 96° C. for3 minutes and at 72° C. for 3 minutes; 35 cycles each at 95° C. for 0.5minute, 52° C. for 0.5 minutes, and 72° C. for 1.5 minutes; 1 cycle at72° C. for 7 minutes; and a soak cycle at 6° C. Taq DNA polymerase wasadded at 72° C. in the first cycle.

PCR reaction products were separated by electrophoresis using a 2%agarose gel (Amresco, Solon, Ohio, USA) with 40 mM Tris base-20 mMsodium acetate-1 mM disodium EDTA (TAE) buffer. A band of approximately600 bp (Fwd and Rev1 primers) and bands of approximately 320 and 370 bp(Fwd and Rev2 primers) were excised from the gel and purified using aMINIELUTE™ Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA)according to the manufacturer's instructions. The purified PCR productswere subsequently analyzed by DNA sequencing. The approximately 600 bpproduct was found to encode a portion of a glycoside hydrolase Family 5polypeptide that was designated CEL5A. The 370 bp product was found toencode a portion of a glycoside hydrolase Family 5 polypeptide that wasdesignated CEL5B.

Example 3 Characterization of the Cel5a Genomic Sequence Encoding theCEL5A Endoglucanase from Penicillium brasilianum IBT 20888

DNA sequencing of the Penicillium brasilianum cel5a PCR product wasperformed with an Applied Biosystems Model 3700 Automated DNA Sequencer(Applied Biosystems, Foster City, Calif., USA) using the Fwd and Rev1primers.

The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence(SEQ ID NO: 2) of the partial Penicillium brasilianum cel5a gene areshown in FIG. 1. Sequences derived from the primer regions used for PCRamplification were excluded from the sequence listing since they do notnecessarily represent the true genomic DNA sequence. The genomic codingsequence of 509 bp encodes a polypeptide of 111 amino acids, interruptedby 3 introns of 67 bp (29-95 bp), 51 bp (249-299 bp), and 56 bp (365-420bp). The % G+C content of the partial gene is 48.1%.

Analysis of the deduced amino acid sequence of the partial cel5a genewith the Interproscan program (Zdobnov and Apweiler, 2001, supra) showedthat the CEL5A polypeptide contained the core sequence typical of aFamily 5 glycoside hydrolase (Interpro domain IPR001547), extending fromapproximately residues 4 to 111 of the predicted polypeptide.

A comparative pairwise global alignment of amino acid sequences inpublic databases was determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, supra) as implemented in the Needle programof EMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the partial Penicillium brasilianum cel5a gene encoding theCEL5A polypeptide shared 72.3% and 72.1% identity (excluding gaps) tothe deduced amino acid sequences of two predicted Family 5 glycosidehydrolase proteins from Aspergillus oryzae and Aspergillus niger,respectively (UniProt accession numbers Q2UPQ4 and O74706,respectively).

Example 4 Characterization of the Cel5b Genomic Sequence Encoding theCEL5B Endoglucanase from Penicillium brasilianum IBT 20888

DNA sequencing of the Penicillium brasilianum cel5b PCR product wasperformed with an Applied Biosystems Model 3700 Automated DNA Sequencer(Applied Biosystems, Foster City, Calif., USA) using the Fwd and Rev2primers.

The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence(SEQ ID NO: 4) of the partial Penicillium brasilianum cel5b gene areshown in FIG. 2. Sequences derived from the primer regions used for PCRamplification were excluded from the sequence listing since they do notnecessarily represent the true genomic DNA sequence. The genomic codingsequence of 299 bp encodes a polypeptide of 73 amino acids, interruptedby 1 intron of 79 bp (179-256 bp). The % G+C content of the partial geneis 54.0%.

Analysis of the deduced amino acid sequence of the partial cel5b genewith the Interproscan program (Zdobnov and Apweiler, 2001, supra) showedthat the CEL5B polypeptide contained the core sequence typical of aFamily 5 glycoside hydrolase (Interpro domain IPR001547), extending fromapproximately residues 10 to 73 of the predicted polypeptide.

A comparative pairwise global alignment of amino acid sequences inpublic databases was determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, supra) as implemented in the Needle programof EMBOSS with gap open penalty of 10, gap extension penalty of 0.5, andthe EBLOSUM62 matrix. The alignment showed that the deduced amino acidsequence of the partial Penicillium brasilianum cel5b gene encoding theCEL5B polypeptide shared 83.6% and 80.1% identity (excluding gaps) tothe deduced amino acid sequences of two predicted Family 5 glycosidehydrolase proteins from Aspergillus fumigatus and Neosartorya fischeri,respectively (UniProt accession numbers Q4X1L7 and A1DGP1,respectively).

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having endoglucanase activity, selected fromthe group consisting of: (a) a polypeptide comprising an amino acidsequence comprising a partial amino acid sequence having at least 75%identity to the partial amino acid sequence of SEQ ID NO: 2 or at least85% identity to the partial amino acid sequence of SEQ ID NO: 4; (b) apolypeptide encoded by a polynucleotide comprising a nucleotide sequencecomprising a partial nucleotide sequence that hybridizes under at leasthigh stringency conditions with (i) the partial nucleotide sequence ofSEQ ID NO: 1 or the partial nucleotide sequence of SEQ ID NO: 3, (ii)the cDNA sequence contained in the partial nucleotide sequence of SEQ IDNO: 1 or the partial nucleotide sequence of SEQ ID NO: 3, or (iii) afull-length complementary strand of (i) or (ii); (c) a polypeptideencoded by a polynucleotide comprising a nucleotide sequence comprisinga partial nucleotide sequence having at least 75% identity to thepartial nucleotide sequence of SEQ ID NO: 1 or at least 85% identity tothe partial nucleotide sequence of SEQ ID NO: 3; and (d) a variantcomprising an amino acid sequence comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of the partialamino acid sequence of SEQ ID NO: 2 or the partial amino acid sequenceof SEQ ID NO: 4.

[2] The polypeptide of paragraph 1, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 75% identity tothe partial amino acid sequence of SEQ ID NO: 2.

[3] The polypeptide of paragraph 2, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 80% identity tothe partial amino acid sequence of SEQ ID NO: 2.

[4] The polypeptide of paragraph 3, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 85% identity tothe partial amino acid sequence of SEQ ID NO: 2.

[5] The polypeptide of paragraph 4, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 90% identity tothe partial amino acid sequence of SEQ ID NO: 2.

[6] The polypeptide of paragraph 5, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 95% identity tothe partial amino acid sequence of SEQ ID NO: 2.

[7] The polypeptide of paragraph 1, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 85% identity tothe partial amino acid sequence of SEQ ID NO: 4.

[8] The polypeptide of paragraph 7, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 90% identity tothe partial amino acid sequence of SEQ ID NO: 4.

[9] The polypeptide of paragraph 8, comprising an amino acid sequencecomprising a partial amino acid sequence having at least 95% identity tothe partial amino acid sequence of SEQ ID NO: 4.

[10] The polypeptide of paragraph 1, comprising an amino acid sequencecomprising the partial amino acid sequence of SEQ ID NO: 2 or thepartial amino acid sequence of SEQ ID NO: 4; or a fragment thereofhaving endoglucanase activity.

[11] The polypeptide of paragraph 10, comprising a amino acid sequencecomprising the partial amino acid sequence of SEQ ID NO: 2 or thepartial amino acid sequence of SEQ ID NO: 4.

[12] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence that hybridizes under at least high stringencyconditions with (i) the partial nucleotide sequence of SEQ ID NO: 1 orthe partial nucleotide sequence of SEQ ID NO: 3, (ii) the cDNA sequencecontained in the partial nucleotide sequence of SEQ ID NO: 1 or thepartial nucleotide sequence of SEQ ID NO: 3, or (iii) a full-lengthcomplementary strand of (i) or (ii).

[13] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 75% identity to the partialnucleotide sequence of SEQ ID NO: 1.

[14] The polypeptide of paragraph 13, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 80% identity to the partialnucleotide sequence of SEQ ID NO: 1.

[15] The polypeptide of paragraph 14, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 85% identity to the partialnucleotide sequence of SEQ ID NO: 1.

[16] The polypeptide of paragraph 15, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 90% identity to the partialnucleotide sequence of SEQ ID NO: 1.

[17] The polypeptide of paragraph 16, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 95% identity to the partialnucleotide sequence e of SEQ ID NO: 1.

[18] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 85% identity to the partialnucleotide sequence of SEQ ID NO: 3.

[19] The polypeptide of paragraph 18, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 90% identity to the partialnucleotide sequence of SEQ ID NO: 3.

[20] The polypeptide of paragraph 19, which is encoded by apolynucleotide comprising a nucleotide sequence comprising a partialnucleotide sequence having at least 95% identity to the partialnucleotide sequence of SEQ ID NO: 3.

[21] The polypeptide of paragraph 1, which is encoded by apolynucleotide comprising a nucleotide sequence comprising the partialnucleotide sequence of SEQ ID NO: 1 or the partial nucleotide sequenceof SEQ ID NO: 3; or a subsequence thereof encoding a fragment havingendoglucanase activity.

[22] The polypeptide of paragraph 21, which is encoded by apolynucleotide comprising a nucleotide sequence comprising the partialnucleotide sequence of SEQ ID NO: 1.

[23] The polypeptide of paragraph 1, wherein the polypeptide is avariant comprising an amino acid sequence comprising a substitution,deletion, and/or insertion of one or more (several) amino acids of thepartial amino acid sequence of SEQ ID NO: 2 or the partial amino acidsequence of SEQ ID NO: 4.

[24] An isolated polynucleotide comprising a nucleotide sequence thatencodes the polypeptide of any of paragraphs 1-23.

[25] The isolated polynucleotide of paragraph 24, comprising anucleotide sequence comprising at least one mutation in the partialnucleotide sequence of SEQ ID NO: 1 or the partial nucleotide sequenceof SEQ ID NO: 3, in which the mutant partial nucleotide sequence encodesthe partial amino acid sequence of SEQ ID NO: 2 or the partial aminoacid sequence of SEQ ID NO: 4.

[26] A nucleic acid construct comprising the polynucleotide of paragraph24 or 25 operably linked to one or more (several) control sequences thatdirect the production of the polypeptide in an expression host.

[27] A recombinant expression vector comprising the nucleic acidconstruct of paragraph 26.

[28] A recombinant host cell comprising the nucleic acid construct ofparagraph 26. [29] A method of producing the polypeptide of any ofparagraphs 1-23, comprising: (a) cultivating a cell, which in itswild-type form produces the polypeptide, under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.

[30] A method of producing the polypeptide of any of paragraphs 1-23,comprising: (a) cultivating a host cell comprising a nucleic acidconstruct comprising a nucleotide sequence encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[31] A method of producing a mutant of a parent cell, comprisingdisrupting or deleting a nucleotide sequence encoding the polypeptide ofany of paragraphs 1-23, which results in the mutant producing less ofthe polypeptide than the parent cell.

[32] A mutant cell produced by the method of paragraph 31.

[33] The mutant cell of paragraph 32, further comprising a gene encodinga native or heterologous protein.

[34] A method of producing a protein, comprising: (a) cultivating themutant cell of paragraph 33 under conditions conducive for production ofthe protein; and (b) recovering the protein.

[35] The isolated polynucleotide of paragraph 24 or 25, obtained by (a)hybridizing a population of DNA under at least high stringencyconditions with (i) the partial nucleotide sequence of SEQ ID NO: 1 orthe partial nucleotide sequence of SEQ ID NO: 3, (ii) the cDNA sequencecontained in the partial nucleotide sequence of SEQ ID NO: 1 or thepartial nucleotide sequence of SEQ ID NO: 3, or (iii) a full-lengthcomplementary strand of (i) or (ii); and (b) isolating the hybridizingpolynucleotide, which encodes a polypeptide having endoglucanaseactivity.

[36] A method of producing a polynucleotide comprising a mutantnucleotide sequence encoding a polypeptide having endoglucanaseactivity, comprising: (a) introducing at least one mutation into thepartial nucleotide sequence of SEQ ID NO: 1 or the partial nucleotidesequence of SEQ ID NO: 3, wherein the mutant nucleotide sequence encodesa polypeptide comprising an amino acid sequence comprising the partialamino acid sequence of SEQ ID NO: 2 or the partial amino acid sequenceof SEQ ID NO: 4; and (b) recovering the polynucleotide comprising themutant nucleotide sequence.

[37] A mutant polynucleotide produced by the method of paragraph 36.

[38] A method of producing a polypeptide, comprising: (a) cultivating acell comprising the mutant polynucleotide of paragraph 37 encoding thepolypeptide under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[39] A method of producing the polypeptide of any of paragraphs 1-23,comprising: (a) cultivating a transgenic plant or a plant cellcomprising a polynucleotide encoding the polypeptide under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.

[40] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-23.

[41] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 24 or 25, whereinoptionally the dsRNA is a siRNA or a miRNA molecule.

[42] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph41, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length. [43] A method of inhibiting the expressionof a polypeptide having endoglucanase activity in a cell, comprisingadministering to the cell or expressing in the cell a double-strandedRNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of thepolynucleotide of paragraph 24 or 25.

[44] The method of paragraph 43, wherein the dsRNA is about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

[45] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with a cellulolytic enzymecomposition in the presence of the polypeptide having endoglucanaseactivity of any of paragraphs 1-23, wherein the presence of thepolypeptide having endoglucanase activity increases the degradation ofcellulosic material compared to the absence of the polypeptide havingendoglucanase activity.

[46] The method of paragraph 45, wherein the cellulosic material ispretreated.

[47] The method of paragraph 45 or 46, wherein the cellulolytic enzymecomposition comprises one or more cellulolytic enzymes are selected fromthe group consisting of a cellulase, endoglucanase, cellobiohydrolase,and beta-glucosidase.

[48] The method of any of paragraphs 45-47, wherein the cellulolyticenzyme composition further comprises a polypeptide having cellulolyticenhancing activity.

[49] The method of any of paragraphs 45-48, wherein the cellulolyticenzyme composition further comprises one or more enzymes selected fromthe group consisting of a hemicellulase, esterase, protease, laccase, orperoxidase.

[50] The method of any of paragraphs 45-49, further comprisingrecovering the degraded cellulosic material.

[51] The method of paragraph 50, wherein the degraded cellulosicmaterial is a sugar.

[52] The method of paragraph 51, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[53] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with a cellulolytic enzymecomposition in the presence of the polypeptide having endoglucanaseactivity of any of paragraphs 1-23, wherein the presence of thepolypeptide having endoglucanase activity increases the degradation ofcellulosic material compared to the absence of the polypeptide havingendoglucanase activity; (b) fermenting the saccharified cellulosicmaterial of step (a) with one or more fermenting microorganisms toproduce the fermentation product; and (c) recovering the fermentationproduct from the fermentation.

[54] The method of paragraph 53, wherein the cellulosic material ispretreated.

[55] The method of paragraph 53 or 54, wherein the cellulolytic enzymecomposition comprises one or more cellulolytic enzymes selected from thegroup consisting of a cellulase, endoglucanase, cellobiohydrolase, andbeta-glucosidase.

[56] The method of any of paragraphs 53-55, wherein the cellulolyticenzyme composition further comprises a polypeptide having cellulolyticenhancing activity.

[57] The method of any of paragraphs 53-56, wherein the cellulolyticenzyme composition further comprises one or more enzymes selected fromthe group consisting of a hemicellulase, esterase, protease, laccase, orperoxidase.

[58] The method of any of paragraphs 53-57, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[59] The method of any of paragraphs 53-58, wherein the fermentationproduct is an alcohol, organic acid, ketone, amino acid, or gas.

[60] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with acellulolytic enzyme composition in the presence of a polypeptide havingendoglucanase activity of any of paragraphs 1-23 and the presence of thepolypeptide having endoglucanase activity increases the degradation ofthe cellulosic material compared to the absence of the polypeptidehaving endoglucanase activity.

[61] The method of paragraph 60, wherein the fermenting of thecellulosic material produces a fermentation product.

[62] The method of paragraph 61, further comprising recovering thefermentation product from the fermentation.

[63] The method of any of paragraphs 60-62, wherein the cellulosicmaterial is pretreated before saccharification.

[64] The method of any of paragraphs 60-63, wherein the cellulolyticenzyme composition comprises one or more cellulolytic enzymes selectedfrom the group consisting of a cellulase, endoglucanase,cellobiohydrolase, and beta-glucosidase.

[65] The method of any of paragraphs 60-64, wherein the cellulolyticenzyme composition further comprises a polypeptide having cellulolyticenhancing activity.

[66] The method of any of paragraphs 60-65, wherein the cellulolyticenzyme composition further comprises one or more enzymes selected fromthe group consisting of a hemicellulase, esterase, protease, laccase, orperoxidase.

[67] The method of any of paragraphs 60-66, wherein the fermentationproduct is an alcohol, organic acid, ketone, amino acid, or gas.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. An isolated polypeptide having endoglucanaseactivity, selected from the group consisting of: (a) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto SEQ ID NO: 4; (b) a polypeptide encoded by a polynucleotidecomprising a nucleotide sequence that hybridizes under at least veryhigh stringency conditions with (i) SEQ ID NO: 3, (ii) a cDNA sequenceof SEQ ID NO: 3, or (iii) a full-length complement of (i) or (ii); and(c) a polypeptide encoded by a polynucleotide comprising a nucleotidesequence having at least 95% sequence identity to SEQ ID NO: 3 or a cDNAsequence thereof.
 2. The polypeptide of claim 1, comprising SEQ ID NO:4; or a fragment thereof having endoglucanase activity.
 3. A nucleicacid construct comprising an isolated polynucleotide comprising anucleotide sequence that encodes the polypeptide of claim 1 operablylinked to one or more heterologous control sequences that direct theproduction of the polypeptide in an expression host.
 4. A recombinantexpression vector comprising an isolated polynucleotide comprising anucleotide sequence that encodes the polypeptide of claim
 1. 5. Anisolated recombinant host cell comprising the nucleic acid construct ofclaim
 3. 6. A method of producing the polypeptide of claim 1,comprising: (a) cultivating an isolated Penicillium brasilianum cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 7. A method of producing the polypeptide ofclaim 1, comprising: (a) cultivating a host cell comprising a nucleicacid construct comprising a nucleotide sequence encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.
 8. A method of producing a mutant of anisolated Penicillium brasilianum parent cell, comprising disrupting ordeleting a nucleotide sequence encoding the polypeptide of claim 1 inthe isolated Penicillium brasilianum parent cell, which results in themutant producing less of the polypeptide than the parent cell.
 9. Amethod of producing the polypeptide of claim 1, comprising: (a)cultivating a transgenic plant or a plant cell comprising apolynucleotide encoding the polypeptide under conditions conducive forproduction of the polypeptide; and (b) recovering the polypeptide.
 10. Atransgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of claim 1, wherein thepolypeptide comprises a signal peptide directing the polypeptide intothe secretory pathway.
 11. A method for degrading a cellulosic material,comprising: treating the cellulosic material with a cellulolytic enzymecomposition in the presence of the polypeptide having endoglucanaseactivity of claim 1, wherein the presence of the polypeptide havingendoglucanase activity increases the degradation of cellulosic materialcompared to the absence of the polypeptide having endoglucanaseactivity.
 12. The method of claim 11, wherein the cellulolytic enzymecomposition comprises one or more cellulolytic enzymes selected from thegroup consisting of an endoglucanase, cellobiohydrolase, andbeta-glucosidase.
 13. The method of claim 11, further comprisingrecovering the degraded cellulosic material.
 14. A method for producinga fermentation product, comprising: (a) saccharifying a cellulosicmaterial with a cellulolytic enzyme composition in the presence of thepolypeptide having endoglucanase activity of claim 1, wherein thepresence of the polypeptide having endoglucanase activity increases thedegradation of cellulosic material compared to the absence of thepolypeptide having endoglucanase activity; (b) fermenting thesaccharified cellulosic material with one or more fermentingmicroorganisms to produce the fermentation product; and (c) recoveringthe fermentation product from the fermentation.
 15. The method of claim14, wherein the cellulolytic enzyme composition comprises one or morecellulolytic enzymes selected from the group consisting of anendoglucanase, cellobiohydrolase, and beta-glucosidase.
 16. The methodof claim 14, wherein steps (a) and (b) are performed simultaneously in asimultaneous saccharification and fermentation.
 17. A method offermenting a cellulosic material, comprising: fermenting the cellulosicmaterial with one or more fermenting microorganisms, wherein thecellulosic material is saccharified with a cellulolytic enzymecomposition in the presence of the polypeptide having endoglucanaseactivity of claim 1 and the presence of the polypeptide havingendoglucanase activity increases the degradation of the cellulosicmaterial compared to the absence of the polypeptide having endoglucanaseactivity.
 18. The method of claim 17, wherein the fermenting of thecellulosic material produces a fermentation product.
 19. The method ofclaim 17, wherein the cellulolytic enzyme composition comprises one ormore cellulolytic enzymes selected from the group consisting of anendoglucanase, cellobiohydrolase, and beta-glucosidase.